Inordinate thrombus formation on blood vessel walls precipitates acute cardiovascular disease states that are the chief cause of death in economically developed societies. Plasma proteins such as fibrinogen, proteases and cellular receptors participating in hemostatis have emerged as important factors that play a role in acute and chronic coronary disease as well as cerebral artery disease by contributing to the formation of thrombus or blood clots that effectively diminish normal blood flow and supply. Vascular aberrations stemming from primary pathologic states such as hypertension, rupture of artheroslerotic plaques or denuded endothelium, activate biochemical cascades that serve to respond and repair the injury site. Thrombin is a key regulatory enzyme in the coagulation cascade; it serves a pluralistic role as both a positive and negative feedback regulator. However, in pathologic conditions the former is amplified through catalytic activation of cofactors required for thrombin generation as well as activation of factor XIII necessary for fibrin cross-linking and stabilization.
In addition to its direct effect on hemostasis, thrombin exerts direct effects on diverse cell types that support and amplify pathogenesis of arterial thrombus disease. The enzyme is the strongest activator of platelets causing them to aggregate and release substances (eg. ADP TXA.sub.2 NE) that further propagate the thrombotic cycle. Platelets in a fibrin mesh comprise the principal framework of a white thrombus. Thrombin also exerts direct effects on endothelial cells causing release of vasoconstrictor substances and translocation of adhesion molecules that become sites for attachment of immune cells. In addition, the enzyme causes mitogenesis of smooth muscle cells and proliferation of fibroblasts. From this analysis, it is apparent that inhibition of thrombin activity constitutes a viable therapeutic approach towards the attenuation of proliferative events associated with thrombosis.
The principal endogeneous neutralizing factor for thrombin activity in mammals is antithrombin III (ATIII), a circulating plasma macroglobulin having low affinity for the enzyme. Heparin exerts clinical efficacy in venous thrombosis by enhancing ATIII/thrombin binding through catalysis. However, heparin also catalyzes inhibition of other proteases in the coagulation cascade and its efficacy in platelet-dependent thrombosis is largely reduced or abrogated due to inaccessibility of thrombus-bound enzyme. Adverse side effects such as thrombocytopenia, osteoporosis and triglyceridemia have been observed following prolonged treatment with heparin.
Hirudin, derived from the glandular secretions of the leech hirido medicinalis is one of the high molecular weight natural anticoagulant protein inhibitors of thrombin activity (Markwardt F. Cardiovascular Drug Reviews, 10, 211, 1992). It is a biopharmaceutical that has demonstrated efficacy in experimental and clinical thrombosis. A potential drawback to the use of Hirudin as a therapeutic agent is likely antigenicity and lack of an effective method of neutralization, especially in view of its extremely tight binding characteristics toward thrombin. The exceedingly high affinity for thrombin is unique and is attributed to a simultaneous interaction with the catalytic site as well as a distal "anion binding exosite" on the enzyme.
Thrombin activity can also be abrogated by Hirudin-like molecules such as hirulog (Maraganore, J. M. et al., Biochemistry, 29, 7095, 1990) or hirutonin peptides (Di Maio, J. et al., J. Med. Chem., 35, 3331, 1992).
Thrombin activity can also be inhibited by low molecular weight compounds that compete with fibrinogen for thrombin's catalytic site, thereby inhibiting proteolysis of that protein or other protein substrates such as the thrombin receptor. A common strategy for designing enzyme inhibitory compounds relies on mimicking the specificity inherent in the primary and secondary structure of the enzyme's natural substrate. Thus, Blomback et al. first designed a thrombin inhibitor that was modeled upon the partial sequence of the fibrinogen A.alpha. chain comprising its proteolytically susceptible region (Blomback, et al., J. Clin. Lab. Invest., 24, 59, 1969). This region of fibrinogen minimally includes the residues commencing with phenylalanine:
Ala-Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly -Gly-Gly-Val-Arg-Gly-Pro-Arg .uparw.scissile bond
Systematic replacement of amino acids within this region has led to optimization of the tripeptidyl inhibitory sequence exemplified by the peptide (D)-Phe-Pro-Arg which corresponds to interactions within the P.sub.3 -P.sub.2 -P.sub.1 local binding sites on thrombin (Bajusz S. et al. in Peptides: Chemistry Structure and Biology: Proceedings of the Fourth American Peptide Symposium, Walter R., Meienhofer J. Eds. Ann Arbor Science Publishers Inc., Ann Arbor, Mich., 1975, pp 603).
Bajusz et al. have also reported related compounds such as (D)Phe-Pro-Arg-(CO)H (GYKI-14166) and (D)MePhe-Pro-Arg-(CO)H (GYKI-14766) (Peptides-Synthesis, Structure and Function: Proceedings of the Seventh American Peptide Symposium, Rich, D. H. & Gross, E. eds., Pierce Chemical Company, 1981, pp. 417). These tripeptidyl aldehydes are effective thrombin inhibitors both in vitro and in vivo. In the case of both GYKI-14166 and GYKI-14766, the aldehyde group is presumed to contribute strongly to inhibitory activity in view of its chemical reactivity toward thrombin's catalytic Ser.sub.195 residue, generating a hemiacetal intermediate.
Related work in the area of thrombin inhibitory activity has exploited the basic recognition binding motif engendered by the tripeptide (D)Phe-Pro-Arg while incorporating various functional or reactive groups in the locus corresponding to the putative scissile bond (i.e. P.sub.1 -P.sub.1 ').
In U.S. Pat. No. 4,318,904, Shaw reports chloromethyl-ketones (PPACK) that are reactive towards Ser.sub.195 and His.sub.57. These two residues comprise part of thrombin's catalytic triad (Bode, W. et al., EMBO Journal 8, 3467, 1989).
Other examples of thrombin inhibitors bearing the (D)Phe-Pro-Arg general motif are those incorporating COOH-terminal boroarginine variants such as boronic acids or boronates (Kettner, C. et al., J. Biol. Chem., 268, 4734, 1993).
Still other congeners of this motif are those bearing phosphonates (Wang, C-L J., Tetrahedron Letters, 33, 7667, 1992) and ###-Keto esters (Iwanowicz, E. J. et al., Bioorganic and Medicinal Chemistry Letters, 12, 1607, 1992).
Neises, B. et al. have described a trichloromethyl ketone thrombin inhibitor (MDL-73756) and Attenburger, J. M. et al. have revealed a related difluoro alkyl amide ketone (Tetrahedron Letters, 32, 7255, 1991).
Maraganore et al. (European 0,333,356; WO 91/02750; U.S. Pat. No. 5,196,404) disclose a series of thrombin inhibitors that incorporate the D-Phe-Pro- moiety and hypothesize that this preferred structure fits well within the groove adjacent to the active site of thrombin. Variations on these inhibitors are essentially linear or cyclic peptides built upon the D-Phe-Pro moiety.
Another series of patents and patent applications have described attempts to develop effective inhibitors against thrombosis by using alpha-ketoamides and peptide aldehyde analogs (EP 0333356; WO 93/15756; WO 93/22344; WO 94/08941; WO 94/17817).
Still others have focused their attention on peptides, peptide derivatives, peptide alcohols, or cyclic peptides as anti-thrombotic agents (WO 93/22344, EP 0276014; EP 0341607; EP 0291982). Others have examines amidine sulfonic acid moieties to achieve this same end (U.S. Pat. No. 4,781,866), while yet others have examined para or meta substituted phenylalanine derivatives (WO 92/08709; WO 92/6549).
A series of Mitsubishi patents and patent applications have disclosed apparently effective argininamide compounds for use as antithrombotic agents. The chemical structures described in these documents represent variations of side groups on the argininamide compound (U.S. Pat. No. 4,173,630; U.S. Pat. No. 4,097,591; CA 1,131,621; U.S. Pat. No. 4,096,255; U.S. Pat. No. 4,046,876; U.S. Pat. No. 4,097,472; CA 2,114,153).
Canadian patent applications 2,076,311 and 2,055,850 disclose cyclic imino derivatives that exhibit inhibitory effects on cellular aggregation.
Many of the examples cited above are convergent by maintaining at least a linear acyclic tripeptidyl motif consisting of an arginyl unit whose basic side chain is required for interaction with a carboxylate group located at the base of the P.sub.1 specificity cleft in thrombin. Two adjacent hydrophobic groups provide additional binding through favourable Van der Waals interactions within a contiguous hydrophobic cleft on the enzyme surface designated the P.sub.3 -P.sub.2 site.
One object of the present invention is to provide thrombin inhibitors that display inhibitory activity towards the target enzyme, thrombin.
A further object of the present invention is to provide thrombin inhibitors that display inhibitory activity towards the target enzyme thrombin and are provided for in a pharmacologically acceptable state.
Still a further object of the present invention is to provide for the use of heterocyclic thrombin inhibitors and formulations thereof as anticoagulant and thrombin inhibitory agents.
Yet a further object of the present invention is to provide for the use of heterocyclic thrombin inhibitors and formulations thereof for therapeutic treatment of various thrombotic maladies.
A further object of the present invention is a process for the synthesis of these low molecular weight thrombin inhibitors. The enzyme inhibitors of the present invention are encompassed by the structure of general Formula I.